A low cost sensitive electrochemical sensor based on functionalized carbon nanotubes and Au nanoparticles deposited on commercial graphite sheets for detection of an antiviral drug, acyclovir (ACV), in pharmaceutical preparations has been developed. The surface morphology of the fabricated electrode was assessed by FT-IR spectroscopy, scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX). This developed sensor exhibited excellent activity for the electrochemical oxidation of ACV. Both CV and linear sweep voltammetry (LSV) were employed to study the electrocatalytic properties of the modified AuNPs/CNTs-COOH/GSE to elucidate the oxidation behavior of ACV. The fabricated sensor exhibited excellent electro-catalytic activity toward ACV determination in pharmaceutical preparations in a wide concentration range of 0.19 μM to 200 μM. Two linear ranges have been obtained for the acyclovir concentration: 0.19–52 μM and 52–200 μM, with a detection limit of 0.007 μM. The lower linear concentration range of 0.19–52 μM showed a sensitivity of 2.18 A M−1 cm−2, and the other one from 52 μM to 200 μM showed a sensitivity of 0.268 A M−1 cm−2. The AuNPs/CNTs-COOH/GSE sensor has been applied for determination of ACV in pharmaceutical preparations. The method presents good sensitivity and stability suitable for routine quantification of drugs in pharmaceutical formulations.